Fermentation process for the production of d-arabitol



' as strains of the genus Zygosaccharomyces.

Unit d t.

FERMENTATION PROCESS FOR THE PRODUC- 'IION F D-ARABITOL No Drawing. Application January 29, 1958 Serial No. 711,802,

Claims priority, application Great Britain February 13, 1957 7 Claims. (Cl. 195-37) This invention relates to the production of polyhydric alcohols, and in particular to the production of D-arabitol, by fermentation.

It is known that certain microorganisms such as the osmophilic yeasts can produce polynydric alcohols such as D-arabitol and glycerol when grown in a fermentation medium containing a high concentration ofglucose. Fermentations of this type are described, for example, in published work by Spencer and Sallans Canadian Journal of Microbiology (1956) 2, pages 72-79. The microorganisms referred to in this work are mainly classified Since some confusion apparently exists between various authorities as to the exact nomenclature of the osmophilic yeasts, the systems of nomenclature adopted in the present application is that described in Lodder and Kreger-van-Rijs book, The Yeasts a Taxonomic Study, published,by Interscience Publishers, New York, 1952. In the nomenclature of these authors all osmophilic yeasts are classified either as Saccharomyces rouxii or Sacch a romyces mllis and it is to these organisms that the present invention is applicable.

Published work has shown that when fermentation tion of one or more of the other alcohols from the samecarbon source.

It is an object of this invention to provide a method of fermentation giving increased yields of D-arabitol.

Accordingly, the present invention is a method for producing D-arabitol, comprising fermenting under submerged and aerated conditions, a suitable nutrient median with a D-arabitol-producing strain of Saccharomyces rouxii or Saccharomyces mellis wherein the nutrient medium contains high test molasses as the major source of assimilable carbon, and recovering D-arabitol from the fermented medium.

The microorganisms are grown under submerged fermentation conditions to provide a liquid fermentation broth which is readily handled in the subsequent recovery stages. Since the fermentation is an aerobic fermentation, aeration is provided, for example by passing air through the medium during fermentation. The passing of ti s; Patsifl air is necessary for the desired fermentation to take I place and mechanical agitation improves the yield of the desired products.

It has been found that certain strains of Saccharomyces rouxii and mellis produce only polyhydric alcohols other than D-arabitol, for example, glycerol and erythritol, and the preent invention is restricted to those strains of the or anisms which produce D-arabitol either with or with 2,934,474 Patented Apr. 26, 1960 out the concomitant production of other polyhydric alcohols.

Suitable strains of the organisms may be selected by the inoculation of a suitable sterile nutrient medium, for example, containing about 20% weight/volume glucose, 1% weight/volume yeast extract and 0.l%-0.l5% weight/volume urea, with the strains of yeast under investigation and subjecting the mixture to shaking at a temperature in the range about 30 C.37 C., for a suitable period, during which samples of the broth are withdrawn and examined by a chromatographic separation on paper to detect the presence of D-arabitol. The chromatographs may be developed, and the polyhydric alcohols detected, by the'method described by Neish, Analytical Methods for Bacterial Fermentations, Second Review, National Research Council of Canada, N.R.C. 2952, 1952. V

By the term a suitable nutrient medium is meant afermentation medium which will support a growth of the micro-organism and the production of D-arabitol during fermentation. In addition to the major carbon source which in the present invention comprises high test'mola'sses, it has been found that the presence of an additionalsimple nitrogen source, such as urea, is required, for example in a concentration of about 0.05% to 0.1% weight for volume to support growth and D- arabitol production. It is also preferred to add a further small proportion of an organic nitrogen source such as corn steep liquor or yeast extract to aid the fermentation, for example, in concentrations of about 0.3% to 1.5%. This substance may also provide a minor proportion of assimilable carbon during the fermentation.

Since .the major proportion of the carbon source is present as high test molasses, it has been found advantageous to add, at the beginning of the fermentation, a small amountof invertase enzyme, for example, about 0.02 to 0.03% volume for volume.- The enzyme is conveniently sterilised with the urea by Seitz filtration in the form of an aqueous solution and then added to remaining ingredients of the medium which have been separately heat sterilised.

The term high test molasses, which is well recognized in the art, includes molasses which are obtained from the extraction of whole sugar cane, and from which no sugar has been crystallised or otherwise extracted for other purposes, such as domestic sugar production. It, therefore, contains substantially the whole of the total sugar content of sugar cane and usually assays at between about 70%. and of total sugars, mainly in the form of sucrose, although some inversion to glucose and fructose may have taken place.

The osmophilic yeasts are capable of growth and fermentation in high concentrations of sugars and it has been found suitable in the process of the present invention to ferment media containing sugar in concentrations in the range of about 15% to 25% weight for volume, although lower or higher concentrations may be fermented if desired.

It has been unexpectedly found that high test molasses when substituted for glucose in the fermentation medium as the maior source of assimilable carbon gives an in crease in the amount of D-arabitol produced during the fermentation. In fermentations when glycerol is produced simultaneously by the organism, it appears, that at least in some ca es. the yield of glycerol is reduced as the vie d of D-arabitol is increased.

D-arabitol may be recovered from the fermented medium by any method known in the art. For example, the

volume of the fermented medium may be reduced by the assesses 'our' copendingapplication 4862/57. Butanol has'been foundto be of particular value-in the recoveryof D-arab-v itol since the desired amount of water may be removed byazeotropic distillation before separating the butanol andall'owing the D-ara'bitol to crystallise out on' cooling. In fermentations which also produce glycerol this compound-"may also-be recovered as a valuahle'by-product, for-example by steam distillation of the fermented mediout before extracting D-arabltol.

QThefollowing example "is-given'to illustrate the increased yield of D-arabitol inafermentationin which the majorsource of assimilalzle carbonis high test molasses when compared-with asimilab-fermentation having an equivalent amount of glucose as a major -source'ofcab "hon.

" Example LTwo' batches .of. fermentation media were prepared {an dheat sterilised.

Medium A contained weightfor volume. or glucose and 1% weight/volume of yeast extract.

. Medium B contained 30% weight for volume of high gestjmolasses (equivalentto 20% weight for volume -total sugars in" the medium) -and0.375 volume f or Volume in cornsteep liquor.

' To medium A was added"O.15% weight/volume urea and'to medium B 0.075% urea which had been sterilised byv Seiti filtration. Also to medium B was added 0.025% invertase enzyme which had been sterilised withithe urea solution. L The media were then seeded .with an inoculum of an o'smophilicstrain 'of Saccharomyces isolated from brood ritnnb pollen and grown in a 20% lucosetmedium in Tishake-flask. "The seeded media were then fermented 'iffor'8 dayswith'aeration at" the rate of 700 mls. oiiair Lbeingexpres'sed on a weight/volume" basis with respect "to the fermentation volume.

D-arabitol, "Glycerol,

percent percent Medium A (20% glucose) 6.2 3. 5 Medium B (30% High Test molasses) 9.0 1. 2

"We claim:

1. A method for producing "D-arabitol, comprising ifermentinglunder submerged and aerated conditionsra =suitable nutrient medium with'a- D-arabitol-producing strain'iof an osmophiliciyeastiselected from the group consisting of Saccharomyces rouxiiz'and Saccharomyces mellis wherein .the. nutrient .medium contains high test molasses as'the major source of-assimilable carbon and recovering D- arabitol from the fermented medium.

2. A method-as claimed in claim lwhereinthe medium also crntains urea.

3. A method as-claimedinclaim ltwherein the medium also contains corn steep liquor.

wi4.Aa'-method asclaimed in claim lwherein the "medi- ,um. also :contains an. extract of yeast. 7

5. A method as claimed in claim 1 wherein the enzyme ':inverta'se..is also .prosentin the nutrient medium.

-A-.method:.as claimedin claim 1 wherein sufficient molassesare presentinthe. medium to provide a concen? .trationof between about 154% to,25% weight for volume ,of total sugars.

7. A methodas .claimedjn claim lwherein the fer- .mentationis carried outat a temperature inrthe range about..3'0IC..to 37 C.

""References Cited in the file of this patent UNITED STATES PATENTS 2.272382 -'Owen FebJlO, 1942 s 2,680,703 Brooks June 8, 1954 .Sencer 'et al. May 28,1957

OTHER: REFERENCES 

1. A METHOD FOR PRODUCING D-ARABITOL, COMPRISING FERMENTING UNDER SUBMERGED AND AERATED CONDITIONS A SUITABLE NUTRIENT MEDIUM WITH A D-ARABITOL-PRODUCING STRAIN OF AN OSMIPHILIC YEAST SELECTED FROM THE GROUP CONSISTING OF SACCHAROMYCES ROUXII AND SACCHAROMYCES MELLIS WHEREIN THE NITRUENT MEDIUM CONTAINS HIGH TEST MOLASSES AS THE MAJOR SOURCE OF ASSIMILABLE CARBON AND RECOVERING D-ARABITOL FROM THE FERMENTED MEDIUM. 